Therapeutic agent for neuropathy in organic acidemia of which mechanism relies on increase in camp

ABSTRACT

[Solving Means] The inventors established technologies for the establishment of iPS cells derived from a methylmalonic acidemia patient and establishment of a stable maintenance and culturing method using peripheral blood lymphocytes of a methylmalonic acidemia patient, and for the differentiation of methylmalonic acidemia patient-derived iPS cells into nerve cells. The inventors made clear that neuropathy in organic acidemia can be treated and prevented by replenishing cAMP using a series of these experiment technologies. The drug of the invention treats or prevents neuropathy by increasing cAMP in organic acidemia.

TECHNICAL FIELD

The present invention relates to a therapeutic agent for neuropathy inorganic acidemia of which mechanism relies on an increase in cAMP.

BACKGROUND ART

Organic acidemia is a disease in which organic acids accumulateexcessively in the living body due to abnormality in metabolizingenzymes. In organic acidemia, due to excessive accumulation of organicacids, metabolic acidosis, hyperammonemia, hypoglycemia, and the likeoccur, and as the results of these, respiration disorder orconsciousness disorder develop in an acute stage, and symptoms such asanorexia and vomiting develop in a chronic stage. Furthermore, organicacidemia is also known such that curative remedy is difficult, and thefatality rate is high.

Examples of the treatment method for organic acidemia include dietetictherapy, pharmacotherapy, dialysis, and implantation.

Dietetic therapy is a method of treatment in which meals with limitedproteins and amino acids such as isoleucine, valine, and methionine,which may be causative substances for organic acid metabolites aretaken. Dietetic therapy enables suppression of accumulation of organicacids, which are causative of organic acidemia; however, dietetictherapy is not capable of completely preventing this accumulation.Therefore, it is an effective method of treatment carried out for thepurpose of stabilizing symptoms in a chronic stage; however, dietetictherapy is not a curative remedy.

Meanwhile, in regard to pharmacotherapy, arginine and CARBAGLU thatdetoxify ammonia, and carnitine hydrochloride that binds to organicacids and accelerates excretion, are used. Pharmacotherapy is a methodof treatment used not only in a chronic stage but also at the time ofaggravation of symptoms in an acute stage. Furthermore, pharmacotherapyis carried out together with hemodialysis in many cases, for the purposeof eliminating organic acids from the blood. Both of thesepharmacotherapy and dialysis are capable of rapid elimination of organicacids at the time of aggravation of symptoms; however, since they do notenable complete exclusion of the accumulation of causative organicacids, these pharmacotherapy and dialysis are not curative remedies.

Compared to these, liver transplantation has been carried out as theonly life supporting method for organic acidemia. Furthermore, since ithas been made obvious that when only liver transplantation is carriedout, neurological symptoms progress slowly, and neurologicalaftereffects and renal disorder come as complications at a high rate,recently, renal and liver co-transplantation of performing livertransplantation and renal transplantation at the same time is carriedout in overseas countries as an advanced treatment.

As a result of enhancement of these transplantation treatment anddialysis technologies, the treatment outcome for organic acidemia hasbeen improved, and the fatality rate has been certainly decreased.However, even with regard to this renal and liver co-transplantation, ithas been reported that the organic acid concentration in thecerebrospinal fluid is not normalized, or severe neuropathy remains.That is, while the fatality rate is decreased by a transplantationtreatment, since neuropathy is not completely cured in survival cases,neuropathy causes deterioration of the QOL of patients.

Under such circumstances, there is a strong demand for the developmentof methods of treatment for neuropathy in organic acidemia.

Meanwhile, cAMP is an intracellular signal transducer functioning as asecond messenger in cells.

The existence, functions and the like of cAMP have been known for long;however, the relevance between cAMP and organic acidemia is not clearlyknown. On the other hand, with regard to cAMP and neuropathy,technologies suggesting this relevance are found here and there (PatentDocuments 1 and 2).

CITATION LIST Patent Document

-   Patent Document 1: WO 2004/045592 A-   Patent Document 2: WO 2007/133749 A

SUMMARY OF THE INVENTION Problem to be Solved by the Invention

In Patent Document 1, a technology related to a composition and a methodfor increasing neurogenesis is disclosed. Furthermore, in PatentDocument 2, a technology related to a method for treating and preventingperipheral neuropathy, and the like is disclosed.

In regard to these related art technologies, regulation of cAMP, a cAMPmodulator, and the like are disclosed, and these suggest the relevancebetween central nervous system damage or peripheral neuropathy and cAMP.

However, even for neuropathy, there may be a variety of causes fordevelopment thereof depending on the disease that brings thisneuropathy, and in these related art technologies, development ofneuropathy in organic acidemia is neither disclosed nor suggested. Inaddition, with regard to neuropathy of organic acidemia, it is thecurrent situation that no treatment based on a mechanism involving cAMPregulation is carried out, and there is no effective therapeutic drug.

Under such circumstances, among organic acidemias, the inventors paidattention to methylmalonic acidemia, which exhibits particularly highfatality rate and is also associated with poor long-term prognosis, andinitiated a study.

The causes of disease of methylmalonic acidemia are roughly classifiedinto deficiency of methylmalonyl CoA mutase (MCM), which is an enzyme,that catalyzes the metabolism from methylmalonyl CoA to succinyl CoA,and metabolic disorder of vitamin B12 (cobalamin), which is a co-enzymeof MCM. It has been considered that due to such deficiency or metabolicdisorder, propionyl CoA and methylmalonyl CoA accumulate in themitochondria, the amount of methylmalonic acid derived from these isincreased, thereby impairing cellular functions, and severeorganopathies are brought about. However, the details of the mechanismhave been not clearly understood.

Under such circumstances, it is an object of the invention to clarifythe mechanism related to neuropathy in methylmalonic acidemia, and todevelop a new therapeutic drug or the like for neuropathy in organicacidemia based on the findings thus obtained.

Means for Solving Problem

The inventors conducted a thorough study, and as the result, they foundthat in organic acidemias including methylmalonic acidemia, a decreasein the cAMP concentration in nerve cells is causative of neuropathy.Thus, the inventors completed inventions such as a drug for treating andpreventing neuropathy in organic acidemia by replenishing cAMP, and thelike.

That is, the inventors conducted an investigation by establishing iPScells from peripheral blood lymphocytes of methylmalonic acidemiapatients and differentiating these cells into nerve cells, and theinventors made clear of the following facts. From such facts, theinventors made clear that neuropathy in organic acidemia can be treatedand prevented by replenishing cAMP.

(1) The inventors discovered six compounds that bring specific recoveryfrom neuronopathy in nerve cells derived from methylmalonic acidemia.

(2) These six compounds all have a common action of increasingintracellular cAMP or activating CREB, which is a downstream molecule.

(3) The cAMP concentration in nerve cells derived from methylmalonicacidemia was measured, and it was confirmed that the concentration wasnoticeably decreased compared to the concentration in nerve cellsderived from a healthy person.

(4) Furthermore, since a decrease in the cAMP concentration was not seenin iPS cells derived from a methylmalonic acidemia patient, it wasconfirmed that this decrease in the cAMP concentration occurs as aresult of differentiation into nerve cells in a methylmalonic acidemiapatient.

(5) In regard to nerve cells derived from methylmalonic acidemia, it wasfound that when cAMP is added into the culture fluid, the cell viabilityand the mitochondrial function are restored to about 90% of those ofnerve cells derived from a healthy person. A similar effect was alsoconfirmed in propionic acidemia.

(6) It was confirmed that in nerve cells derived from methylmalonicacidemia, a decrease in cAMP is caused by a decrease in the activity ofAdenylate cyclase. In addition, the activity of CREB that uses cAMP as asubstrate is not inhibited, and when Adenylate cyclase-derived cAMP issupplemented, CREB is phosphorylated. Thus, it was confirmed that atherapeutic effect is exhibited on the basis of this phosphorylation asone of the mechanisms.

(7) In an in vivo model produced through administration of methylmalonicacid, an effect of suppressing the number of times and duration ofseizure caused by convulsion and an effect of increasing the survivalrate were confirmed, by administering a drug that supplements cAMP tothe in vivo model.

The invention has the following configurations.

A first configuration of the invention is a therapeutic drug forneuropathy, intended for treating or preventing neuropathy by increasingcAMP in organic acidemia.

A second configuration of the invention is the therapeutic drug forneuropathy according to the first configuration, further intended foractivating CREB.

A third configuration of the invention is the therapeutic drug forneuropathy according to the first or second configuration, in which theorganic acidemia is an organic acidemia lowering the activity ofAdenylate cyclase.

A fourth configuration of the invention is the therapeutic drug forneuropathy according to the first or second configuration, in which theorganic acidemia is selected from any one or a plurality ofmethylmalonic acidemia and propionic acidemia.

A fifth configuration of the invention is the therapeutic drug forneuropathy according to any one of the first to fourth configurations,in which the increase in cAMP is achieved by any one of Forskolin,GW9508, NECA, SKF77434, nicotinamide, dobutamine, and db-cAMP.

A sixth configuration of the invention is the therapeutic drug forneuropathy according to the third configuration, in which the increasein cAMP is achieved by suppressing or preventing a decrease in theactivity of Adenylate cyclase.

A seventh configuration of the invention is the therapeutic drug forneuropathy according to any one of the first to sixth configurations, inwhich a life prolongation effect is increased by treating or preventingneuropathy.

An eighth configuration of the invention is a method for treating orpreventing neuropathy in organic acidemia by using the therapeutic drugfor neuropathy according to any one of the first to seventhconfiguration.

Effect of the Invention

According to the invention, the mechanism related to neuropathy inmethylmalonic acidemia has been made clear, and also, a new therapeuticdrug or the like for neuropathy in organic acidemia based on thisfinding can be provided.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a diagram showing an investigation of the suitability of ascreening experiment system using a graph.

FIG. 2 shows a protective action of dibutyl-cAMP for neuronal precursorcells derived from a methylmalonic acidemia patient.

FIG. 3 is a diagram showing a comparison of the intracellular cAMPconcentrations in normal iPS cell-derived neuronal precursor cells(201B7 NPCs) and methylmalonic acid patient iPS cell-derived neuronalprecursor cells (B47 NPCs).

FIG. 4 is a diagram showing a comparison of the intracellular cAMPproduction abilities depending on the addition of Forskolin.

FIG. 5 is a diagram showing an investigation of the influence of theaddition of methylmalonic acid on the intracellular cAMP concentrationin methylmalonic acid patient iPS cell-derived neuronal precursor cells(B42 NPCs).

FIG. 6 is a diagram showing an investigation of the amount of expressionof various proteins under various conditions in methylmalonic acidpatient iPS cell-derived neuronal precursor cells (B42 NPCs).

FIG. 7 is a diagram showing the results of comparing and investigatingthe numbers of times of seizure measured before and after theadministration of a candidate drug for cAMP increase in a simplifiedneuropathy model of methylmalonic acidemia.

FIG. 8 is a diagram showing the results of comparing and investigatingthe total durations of seizure measured before and after theadministration of a candidate drug for cAMP increase in a simplifiedneuropathy model of methylmalonic acidemia.

FIG. 9 is a diagram showing the results of comparing and investigatingthe survival rates in the presence or absence of administration of acandidate drug for cAMP increase in a lethal methylmalonic acid seizuremodel.

MODE(S) FOR CARRYING OUT THE INVENTION

A therapeutic drug for neuropathy and the like of the invention will beexplained.

The inventors achieved, for the first time, experiment technologies forthe establishment of iPS cells derived from a methylmalonic acidemiapatient and establishment of a stable maintenance and culturing methodusing peripheral blood lymphocytes of a methylmalonic acidemia patient,and for the differentiation of methylmalonic acidemia patient-derivediPS cells into nerve cells.

The inventors made clear of the following facts by using a series ofthese experiment technologies. From such facts, the inventors clarifiedthat neuropathy in organic acidemia can be treated and prevented bysupplementing cAMP.

(1) The inventors discovered six compounds that bring specific recoveryfrom neuronopathy in nerve cells derived from methylmalonic acidemia.

(2) These six compounds all had a common action of increasingintercellular cAMP or activating CREB, which is a downstream molecule.

(3) The cAMP concentration in nerve cells derived from methylmalonicacidemia was measured, and it was confirmed that the concentration wasnoticeably decreased compared to the concentration in nerve cellsderived from a healthy person.

(4) Furthermore, since a decrease in the cAMP concentration was not seenin iPS cells derived from a methylmalonic acidemia patient, it wasconfirmed that this decrease in the cAMP concentration occurs as aresult of differentiation into nerve cells in a methylmalonic acidemiapatient.

(5) In regard to nerve cells derived from methylmalonic acidemia, it wasfound that when cAMP is added into the culture fluid, the cell viabilityand the mitochondrial functions are restored to about 90% of those ofnerve cells derived from a healthy person. A similar effect was alsoconfirmed in propionic acidemia.

(6) In regard to nerve cells derived from methylmalonic acidemia, it wasconfirmed that a decrease in cAMP is caused by a decrease in theactivity of Adenylate cyclase. In addition, it was confirmed that theactivity of CREB that uses cAMP as a substrate is not inhibited, andwhen Adenylate cyclase-derived cAMP is supplemented, CREB isphosphorylated. Thus, it was confirmed that a therapeutic effect isexhibited on the basis of this phosphorylation as one of the mechanisms.

(7) In an in vivo model produced through administration of methylmalonicacid, an effect of suppressing the number of times and duration ofseizure caused by convulsion and an effect of increasing the survivalrate were confirmed, by administering a drug that supplements cAMP tothe in vivo model.

The therapeutic drug for neuropathy of the invention treats or preventsneuropathy in organic acidemia by increasing cAMP. Furthermore, as anembodiment for the treatment of neuropathy, for example, a decrease inthe number of times of convulsive seizure, a decrease in the duration ofconvulsion, and an increase in the life prolongation effect may bementioned.

Organic acidemia is defined as a disease in which organic acids areexcessively accumulate in the living body due to abnormality ofmetabolic enzymes, and typical examples include methylmalonic acidemiaand propionic acidemia.

The therapeutic drug for neuropathy according to the invention isdefined as a compound that can achieve an increase in cAMP at aneuropathic lesion in organic acidemia, or a composition including thiscompound as an active ingredient. Furthermore, in view of the purpose ofthe invention, such a compound may be used with an intact chemicalstructure to achieve an increase in cAMP, or the compound can beconverted to a so-called DDS-formulated compound, which receivesmetabolism or the like in the living body to have the structure of thecompound changed, and thereby achieves an increase in cAMP at a lesion.Regarding such a compound, for example, db-cAMP, which is a compoundhaving increased cell permeability of cAMP, can be used.

The therapeutic drug for neuropathy of the invention may furtheractivate cAMP response element binding protein (CREB) in addition to anincrease in cAMP. Since a drug that is more suitable for the mechanismof neuropathy in organic acidemia can be obtained thereby, anenhancement of the therapeutic effect for neuropathy can be expected.Examples of such a compound include Forskolin ((3R,4aR,5S,6S,6aS,10S,10aR,10bS)-6,10,10b-trihydroxy-3,4a,7,7,10a-pentamethyl-1-oxo-3-vinyldodecahydro-1H-benzo[f]chromen-5-ylacetate), GW9508 (3-(4-(((3-(Phenoxy) phenyl) methyl) amino) phenyl)propanoic acid), NECA (5′-N-Ethylcarboxamidoadenosine1-(6-Amino-9H-purin-9-yl)-1-deoxy-N-ethyl-β-D-ribofuranuronamide),SKF77434 (3-allyl-1-phenyl-1,2,4,5-tetrahydro-3-benzazepine-7,8-diol),nicotinamide (pyridine-3-carboxamide), and dobutamine.

It is not necessary to particularly limit the dosage form of thetherapeutic drug for neuropathy of the invention, and various dosageforms can be used. Typically, a form of a preparation for oralingestion, such as a tablet, a capsule, or a powder, can be adopted, andin addition to that, a form of an injectable preparation, a transdermalabsorption type preparation or the like can be adopted.

EXAMPLES I. Experiment Materials and Experiment Method

<1. Method for Regulating Patient-Derived Peripheral Blood Lymphocytes>

(1) For patients, patients of organic acidemia (methylmalonic acidemiaand propionic acidemia), which is the object of the present study, wereselected as subjects from among ambulatory diagnostic patients of thepediatrics department of Kumamoto University Hospital. Neonatal severecases already confirmed from enzymatic diagnoses and gene diagnoses wereselected as subjects. Regarding the explanation and consent of subjectpatients, a written consent approved by the ethics committee in KumamotoUniversity Life Sciences Research Department was used.

(2) From those patients whose consent was obtained, 3 mL of patientblood was collected in the pediatrics ambulatory treatment room. Thisblood was aseptically transferred into a blood collecting tube (CPT) forVACUTAINER lymphocyte separation (BD catalogue No. 362753) and was movedto the fifth floor of the medical department clinical study building.

(3) The blood collecting tube for VACUTAINER lymphocyte separation (CPT)was centrifuged at 20° C. and 2,000 g/30 minutes, and a mononuclear celllayer was suctioned and then aseptically moved to into a fresh asepticSpitz tube in a biohood.

(4) 20 mL of an aseptic phosphate buffer solution was added to the freshaseptic Spitz tube, the mixture was stirred and centrifuged at 2,000g/20 minutes, and then the supernatant was removed.

(5) Mononuclear cell pellets were suspended in 500 μL of BAMBANKER hRM(catalogue No. CS-07-001), and then the suspension was preserved at −80°C.

<2. Method for Establishing Patient-Derived iPS Cells>

(1) The preserved monocytic cells were stored in liquid nitrogen andthen were transported to the building of Kumamoto University Instituteof Molecular Embryology and Genetic.

(2) The preserved tube was melted in a constant temperature chamber at37° C. Before the preserved tube was completely melted, 9.5 mL of D-PBSwas added thereto, and the mixture was stirred.

(3) After melting, the mixture was centrifuged at 100×g/10 minutes at10° C., and the supernatant was removed. Furthermore, 12 mL of D-PBS wasadded to the residue, the mixture was centrifuged at 100×g/10 minutes at18° C., and the supernatant was removed.

(4) 5 mL of mononuclear cell medium (PBMC-CM: 100 mL of PBMC medium+100mL of SCF stock solution+100 mL of FLT-3 stock solution+100 mL of TPOstock solution+IL6 stock solution) was added to the residue, and theresidue was suspended. For the suspension, the number of live cells wascounted by Trypan Blue staining.

(5) The cells were suspended using PBMC-CM medium at a proportion of5×10⁵ cells/well, and the suspension was inoculated onto a 24-well plateat a concentration of 0.5 mL/well. After the inoculation, culture wascarried out in an incubator at 37° C. and 5% CO₂, and from the next day,medium exchange was carried out consecutively for 3 days.

(6) Trypsin was added to a well for counting the number of cells, thecells were detached, and the number of cells was counted.

(7) A vector solution equivalent to MOI=5 was produced in PBMC-C medium,and the solution was added to each of the wells. For two days after theinfection, the medium was exchanged everyday.

(8) On the second day after infection, mitomycin-treated mouse embryofibroblasts (MEF) were inoculated onto a 6-well plate to a concentrationof 1×10⁶ cells/well and were cultured at 37° C. and 5% CO₂.

(9) On the third day after infection, the infected cells were detachedby pipetting and were superposed on MEF prepared on a 6-well plate(1×10⁵ cells/well), and the cells were cultured in an incubator at 37°C. and 5% CO₂.

(10) From the fourth day to the sixth day after infection, an entiremedium exchange was carried out with PBMC medium every day.

(11) On the seventh day after infection, 1 mL of the medium from 2 mLper well of the 6-well plate, and 1 mL of human iPS medium (500 mL ofDMEM/F12+5 mL of NEAA+6.25 mL of 200 mM L-Glutamine+125 mL of KSR+500 μLof 0.1 M 2-Mercaptoethanol/PBS+5 ng/mL rhbFGF) was added to each well.Culture was continued in an incubator at 37° C. and 5% CO₂.

(12) From the eighth day to the twenty-first day after infection, anl-carnitine solution (L-cartine FF injection) was added to human iPSmedium to a concentration of 500 nM, and the entire medium exchange wascarried out using this FFiPS medium everyday, while observations weremade twice a day.

(13) iPS colonies were picked up, and fifty clones in total wereestablished. The patient-derived iPS cells thus established weresubsequently used as disease-derived iPS cells.

(14) The clones thus established were all checked for positivity usingalkaline phosphatase (AP100R-1), Nanog (SAB-103A-1), OCT4 (SAB-105A-1),TRA1-60 (SAB-100A-1), and SSEA-3 (SAB 102A-1). Furthermore, in all ofthe clones thus established, expression of a human iPS cellundifferentiated marker was confirmed (Human iPS cell indentificationprimer set ABP SC IPSHRES).

<3. Method for Maintaining and Culturing Disease-Derived iPS Cells>

The disease-specific iPS cells thus established had weak cellproliferation power and were not apt for maintenance and culture.Therefore, carnitine that has been reported to partially restore themitochondrial functions of the present disease was added to the standardculture fluid, and culture was carried out. Thus, it was newly foundthat maintenance and culture were possible. Therefore, maintenance andculture were enabled using the following culturing method.

(1) According to the day of subculturing, culture of mitomycin C-treatedMEF (3×10⁵ cells/60 mm well) was carried out in advance.

(2) For disease-derived iPS cells, wells having sufficiently enlargedcolonies were selected and washed two times with PBS. A celldissociation solution (0.25 g of Trypsin, 10 mg/mL Collagenase IV, 20 mLof KSR, 100 μL of 1 M CaCl₂/PBS, and 70 mL of PBS) was added at aconcentration of 500 μL/60 mm well, and the cells were incubated for 5minutes in an incubator at 37° C. and 5% CO₂.

(3) 2 mL of a medium was added to detach the cells. 15 mL of the cellsuspension was transferred into a conical tube, 5 mL of a medium wasadded thereto, and the mixture was centrifuged at 170×g/5 minutes.

(4) The supernatant was suctioned, and the residue was suspended infresh FFiPS medium (carnitine-added medium). The suspension wasinoculated onto MEF, and culture was carried out in an incubator at 37°C. and 5% CO2.

(5) Any undifferentiated marker was checked in every five subcultures.

<4. Method for Inducing Disease-Derived iPS Cells into NeuronalPrecursor Cells>

Induction of nerve cells appropriate for high-throughput drug discoveryscreening was attempted by various kinds of methods. As the result, itwas found that the following method is suitable.

(1) For the induction of differentiation, a GIBCO Neural InductionMethod was used. Regarding the initial induction, Neural InductionMedium (NIM: 490 mL of Neuralobasal Medium+10 mL of GIBCO NeuralInduction Supplement) was used. Furthermore, as the maintenance culturefluid, Neural Expansion Medium (NEM: 49 mL of Neurobasal medium+49 mL ofAdvanced DMEN/F12+2 mL of GIBCO Neural Induction Supplement) was used.

(2) 70% to 80% confluent iPS cells were used for the induction ofdifferentiation.

(3) The culture fluid after a lapse of 24 hours was suctioned, and cellsthat did not adhere were all removed. After this operation, NIM culturefluid was added thereto, and culturing was continued. The culture fluidwas exchanged after every 48 hours.

(4) NSC(P) near the seventh day was subcultured as follows.

-   -   First, a culture plate coated with Geltrex matrix was prepared,        and NIM was removed from the culture plate.    -   After the removal, the culture plate was washed with 2 mL of        PBS. After washing, 1 mL of a dissociation solution was added,        and the culture plate was incubated for 5 minutes.    -   The dissociation solution was collected in a 15-mL conical tube,        washing-up of the culture plate was performed with 1 mL of PBS,        and the PBS was collected into the conical tube. After the        collection, the collected liquid was pipetted three times with a        5-mL pipette.    -   After pipetting, the cell solution was filtered through a 100-μm        cell strainer (FALCON). The cells thus filtered were centrifuged        for 4 minutes at 300 g. The supernatant was suctioned, the        residue was suspended in PBS in an amount of 3 mL×well, and the        suspension was centrifuged at 300×g for 4 minutes. Furthermore,        the supernatant was suctioned, and the residue was suspended in        NEM.    -   The cells were inoculated so as to obtain a concentration of        from 2×10⁵ cells/mL to 4×10⁵ cells/mL. Furthermore, Y27632 was        added to the cell suspension to a concentration of 5 μM.    -   After 24 hours, the culture fluid was removed, and the culture        fluid was exchanged with fresh NEM culture fluid.

(5) For the subsequent screening process, this Neural Progenitor cells(NPCs) were used.

II. Establishment of Screening System

<1. Drug Screening Candidate Compound List>

In the present screening experiment, a compound library (trade name:SIGMA PETIT SCREENING 2014), commercially available from Sigma-AldrichCorporation, which consists of 440 kinds of compounds, the operatingmechanisms of which have been identified, was used. The compound libraryused for the present Experiment Example is shown in the following table.

TABLE 1 CAT. NO. Number of PETIT SCREENING 2014 Series (compound list)compounds Naturally sorted IV S990043-NAT4 80 active substance library(old version) Cancer-suppressing I S990043-ATU1 80 compound library IIS990043-ATU2 80 Agonist/activated I S990043-AGO1 80 factor library IIS990043-AGO2 40 Inhibitor library IV S990043-INH4 80 V S990043-INH5 80VI S990043-INH6 80 VII S990043-INH7 80

<2. Screening Method>

[Screening System 1: Screening System for Compounds that Reduce ExogenicMethylmalonic Acid and Propionic Acid Toxicity Using Skin Fibroblasts]

(1) Fibroblasts derived from a healthy child and a patient wereprecultured. The cultured cells were inoculated, in a 80% confluentstate, onto a 96-well collagen-coated dish at a proportion of 5×10³cells/well. After the inoculation, the cells were precultured in anincubator at 5% CO₂ for 24 hours.

(2) A methylmalonic acid solution at a concentration of 200 μM was addedto DMEM/F12/10% FBS medium, and culturing was carried out. Regardingpropionic acid, a similar operation was carried out by adjusting theconcentration to 400 M.

(3) Each compound was added to each well at a concentration of 5 M. Asan object of comparison, DMSO was added.

(4) After 24 hours, measurement of the light absorbance through a 450-nmfilter was carried out using the WST-8 method.

[Screening System: Screening System for Compounds that AmeliorateEndogenic Metabolic Disturbance Using Skin Fibroblasts]

(1) Screening was carried out by a method similar to that of thescreening system 1. That is, fibroblasts derived from a healthy childand a patient were precultured. The cultured cells were inoculated, in a80% confluent state, onto a 96-well collagen-coated dish at a proportionof 5×10³ cells/well. After the inoculation, the cells were preculturedin an incubator at 5% CO₂ for 24 hours.

(2) Each compound was added to each well at a concentration of 5 μM.

(3) After 24 hours, measurement of the light absorbance through a 450-nmfilter was carried out using the WST-8 method.

[Screening System 3: Screening System for Therapeutic Compounds thatReduce Exogenic Neurotoxicity Using Neuronal Precursor Cells (NPCs)Induced from Disease-Specific iPS Cells]

(1) NPCs were cultured using the above-described differentiationinduction method (1-3).

(2) NPCs were inoculated onto a 96-well geltrex-coated dish (GIBCO) soas to obtain a concentration of 1×10⁵ cells/well. After the inoculation,culturing was carried out in an incubator at 5% CO₂ at 37° C. for 24hours.

(3) After 24 hours, methylmalonic acid was added to the culture fluid soas to obtain a concentration of 100 μM. In the case of propionic acid,the acid was added to the culture fluid supernatant so as to obtain aconcentration of 200 μM.

(4) Each compound was added to each well so as to obtain a concentrationof 5 μM. As an object of comparison, DMSO was added.

(5) After 24 hours, measurement of the light absorbance was carried outusing the WST-8 method.

[Screening System 4: Screening System for Compounds that AmeliorateEndogenic Metabolic Function Disturbance Using Neuronal Precursor Cells(NPCs) Induced from Disease-Specific iPS Cells]

(1) Screening was carried out by a method similar to that for thescreening system 3. That is, NPCs were cultured using theabove-described differentiation induction method (1-3).

(2) NPCs were inoculated onto a 96-well geltrex-coated dish (GIBCO) soas to obtain a concentration of 1×10⁵ cells/well. After the inoculation,culturing was carried out in an incubator at 5% CO₂ at 37° C. for 24hours.

(3) Each compound was added to each well so as to obtain a concentrationof 5 M. As an object of comparison, DMSO was added.

(4) After 24 hours, measurement of the light absorbance was carried outusing the WST-8 method.

<3. Validation Results for Screening Systems>

(1) Validity of the present screening systems was verified.

(2) In the screening experiment, an investigation is carried out using a96-well plate, and the purpose of the screening experiment is to screena compound that reduces organic acid toxicity. From this point of view,the 0% control of the first and twelfth wells were made into wells towhich methylmalonic acid (or propionic acid) was not added, and thesecond to the eleventh wells were used for a toxicity test. The resultsare shown in Table 2 and FIG. 1.

(3) From Table 2, the following were obtained: CV value <10%, S/Bratio >3, S/N ratio >80, and Z-factor >0.5. Thus, the screening systemswere considered as adequate screening systems.

(4) Therefore, the present screening systems were used for the screeningof compounds in the subsequent Experiment Examples, as appropriateexperiment systems.

TABLE 2 1 2 3 4 5 6 7 8 9 10 11 12 A 359 2125 2149 2047 2327 2021 20892126 2219 2216 2220 427 B 441 1995 2006 2123 2002 2195 2155 2014 20612219 3144 415 C 401 2215 2135 2145 2008 2120 2220 2245 2065 2152 2195998 D 379 1998 2250 2320 2228 2215 2219 2214 2225 2265 2189 389 E 4052010 2106 2105 2215 2213 2096 2210 2216 2220 2058 389 F 411 2108 22052114 2118 2258 2230 2035 2115 2118 2207 419 G 423 2250 2280 2350 22202214 2268 2265 2197 2305 2136 413 H 415 2215 2235 2285 2224 2218 22372267 2236 2360 2189 367 Average 404 2115 2171 2186 2168 2182 2189 21712167 2232 2160 402 SD 25.52 105.21 89.92 114.25 115.01 75.67 67.44100.18 74.14 78.16 52.58 19.94 Z-factor 0.82 CV 4.0814 S/B 5.4061 S/N88.862

III. Experiment Example Experiment Example 1. Influence of VariousCompounds on Neuronopathy

(1) An investigation was conducted using the various compounds shown inTable 1 to find what influence the various compounds would have onneuronopathy.

(2) The results are presented in Table 3.

-   -   To explain an outline of Table 3, a statistical treatment was        carried out using a heat map, and an average of the negative        controls (first row and twelfth row of 96 wells) was determined        and designated as −1, while an average of the positive controls        (not shown in the diagram) was determined and designated as +1.        Each of the drug data was subjected to normalization into a        value between −1 and +1, and the experiment was repeated three        or more times. Drug data exhibiting high reproducibility and        efficacy are surrounded by ◯.    -   From these results, in cells derived from a patient, six kinds        of compounds enabled recovery from neuronopathy in a        disease-specific manner. These compounds were Forskolin, GW9508,        NECA, SKF77434, nicotinamide, and dobutamine.

(3) Furthermore, it was found that all of these compounds have a commonaction of increasing intracellular cAMP and having an effect ofactivating CREB, which is a downstream key regulator.

Experimental Example 2. Secondary Screening Using Patient-DerivedNeuronal Precursor Cells

(1) From the results of Experiment Example 1, a possibility that cAMPwould exhibit a therapeutic effect on methylmalonic acidemia wassuggested. From this point of view, an investigation was conducted tosee whether cAMP has a protective effect on methylmalonic acidemiapatient-derived neuronal precursor cells (hereinafter, MMApatient-derived neuronal precursor cells).

(2) MMA patient-derived neuronal precursor cells were cultured for 24hours on a 96-well plate (Gelatin-coated) at a concentration of 1×10⁴cells/well, db-cAMP was added at a concentration of 0, 500 nM, 1 μM, 10μM, or 500 M, and the cells were cultured for 24 hours. Subsequently,the light absorbance was measured by the WST8 method.

(3) The results are shown in FIG. 2. A cell protecting effect wasrecognized proportionally to the concentration of cAMP added to theculture supernatant. It was found that this effect restores to about 90%of a normal subject at the maximum.

Experiment Example 3. Experiment for cAMP Comparison BetweenPatient-Derived Nerve Cells and Normal Nerve Cells

(1) From the results of Experiment Example 1 and Experiment Example 2, apossibility that a disease-specific decrease in the intracellular cAMPconcentration may be a disease condition of the present disease.Therefore, for the purpose of investigating whether a cell protectiveeffect of cAMP on nerve cells derived from methylmalonic acidemia isspecific to methylmalonic acidemia, the intracellular cAMP concentrationwas measured.

(2) For the measurement of the intracellular cAMP concentration, a cAMPcomplete ELISA kit (ADI-900-163) of Enzo Co., Ltd. was used.

(3) On the occasion of performing measurement, a pretreatment of cellswas carried out.

-   -   Cells were detached from the culture plate, the number of cells        was counted, and centrifugation was performed. After the        centrifugation, the supernatant was suctioned, and the residue        was suspended in a 0.1 M HCl solution at a concentration of        1×10⁶/cells/mL. In the case of cells with a known cell count, a        series of these operations was omitted, and 0.1 M HCl was        introduced directly onto the culture plate while the cells were        adhered.    -   After suspending, the suspension was incubated at room        temperature for 10 minutes and then was centrifuged at 600 g/5        minutes. This supernatant was preserved at −80° until the        measurement of cAMP was performed.

(4) For the measurement, measurement was carried out through a series ofthe following processes.

-   -   A neutralizing liquid (Neutralizing reagent) was introduced in        an amount of 50 L into wells other than total activity (TA) and        blanks.    -   Subsequently, 100 μL of a standard diluent was added to        non-specific binding (NBS) and BO (0 pmol/mL standard).        Furthermore, 50 μL of a standard diluent was added to the wells        of NBS. Furthermore, 100 μL each of reference dilution series        from #1 to #5 was added.    -   A sample was added to each well in an amount of 100 μL each.    -   A blue conjugate solution was introduced in an amount of 50 μL        each into wells other than total activity (TA) and blanks.    -   Yellow (yellow antibody) was introduced in an amount of 50 μL        each into wells other than blanks, TA, and NSB (blank:        transparent, TA: transparent, NSB: blue, sample and standard        were green).    -   The plate was sealed and was incubated for 2 hours at room        temperature in a shaker.    -   After incubation, all wells were suctioned, and then the plate        was washed three times in total with 400 μL of a washing        solution. Finally, the plate was dried on a KIMTOWEL.    -   5 μL of a blue (blue conjugate) solution was added to the wells        of TA.    -   200 μL of a substrate solution was added, the plate was left to        stand for one hour at room temperature, and then 50 μL of a stop        solution was added thereto. The light absorbance at 405 nm was        measured.

(5) The results are presented in FIG. 3. In FIG. 3, 201B7 NPCs representthe intracellular cAMP concentration of normal iPS cell-derived neuronalprecursor cells, and B42NPCs represent the intracellular cAMPconcentration of methylmalonic acid patient iPS cell-derived neuronalprecursor cells.

(6) A comparison between the cAMP concentration in the methylmalonicacid-derived nerve cells and the cAMP concentration in the normal nervecells was carried out, and as a result, it was found that the cAMPconcentration was noticeably decreased in the methylmalonic acid-derivednerve cells.

(7) As a result of a series of experiments carried out thus far, it wasfound that the cAMP concentration was decreased in the methylmalonicacidemia patient-derived neuronal precursor cells, and the cellularfunctions were restored by replenishing cAMP. This suggests apossibility that a decrease in cAMP represents a disease state specificto the present disease, and a treatment of replenishing cAMP may becomea fundamental treatment.

Experiment Example 4. cAMP Production Ability Test for MethylmalonicAcidemia Patient

(1) The cAMP production ability in methylmalonic acidemiapatient-derived neuronal precursor cells was measured.

(2) Specifically, Neural Progenitor cells (NeuP) were induced fromnormal iPS cells and MMA-derived iPS cells. To these cells, Forskorin(FSK) was added at a concentration of 10 μmol/l, and the intracellularcAMP concentration after 24 hours was measured.

(3) The results are presented in FIG. 4. As a result of theinvestigation, in methylmalonic acidemia-derived NPCs, a decrease inreactivity to Forskorin was recognized, and it was suggested that thecAMP production ability was deteriorated.

Experiment 5. Signal Inhibition Test

(1) Signal transduction involving cAMP is well known, and an inhibitionexperiment of using a library of drugs that inhibit these pathways wascarried out. Raw data of the results are presented in Table 4.

(2) As the result, the following were found.

-   -   A cAMP production inhibitor aggravated cell damage of        methylmalonic acid.    -   A cAMP decomposition inhibitor ameliorated cell damage of        methylmalonic acid.    -   ERK inhibition aggravated cell damage of methylmalonic acid.    -   CREB inhibition ameliorated cell damage of methylmalonic acid.

(3) Furthermore, from an experiment of using agonists and inhibitors,the following were found.

-   -   When ERK is activated, and CREB is inhibited, cytotoxicity is        exacerbated.    -   Even if cAMP is added in a state of having CREB inhibited, a        cell protecting effect is not obtained.

(4) From the above results, it was found that cell damage (toxicity) ofmethylmalonic acid is caused by decreased production of intracellularcAMP, this is cellular dysfunction caused by chronic deterioration ofthe production ability of cAMP, and a therapeutic effect of db-cAMP isexhibited through CREB.

Experiment 6. Experiment on Adenylate Cyclase Inhibition byMethylmalonic Acid

cAMP is produced from ATP by Adenylate cyclase. From this point of view,an investigation was conducted on whether methylmalonic acid wouldinhibit Adenylate cyclase.

1. With regard to methylmalonic acidemia patient iPS cell-derivedneuronal precursor cells (B42NPCs), culturing was carried out with aculture fluid to which methylmalonic acid had been added to a finalconcentration of 50 μM or 100 μM, and the cAMP concentration after 24hours was measured.

2. The results are shown in FIG. 5 and FIG. 6. Meanwhile, the proteinexpression analysis shown in FIG. 6 was carried out based on theexisting Western Blotting method.

(1) It was found that as the methylmalonic acid concentration in theculture fluid is higher, the cAMP production power is decreased (FIG.5).

(2) On the other hand, the total amounts of protein expression ofAdenylate cyclase and CREB were constant irrespective of the presence orabsence of the addition of methylmalonic acid (FIG. 6).

(3) Furthermore, it was found that even in the presence of MMA, in acase in which cAMP was added, the expression of phosphorylated CREB wasincreased, and phosphorylation (activation) of CREB was not inhibited.

(4) From these, it was found that a decrease in the cAMP productioncaused by MMA addition is attributed to a decrease in the activity ofAdenylate cyclase.

From the above results, the inventors found that cell damage (toxicity)of methylmalonic acid is caused as the intracellular cAMP concentrationis decreased by a decrease in the activity of Adenylate cyclase. Inaddition, it was found that such mechanism causes cellular dysfunctioncaused by a decrease in the chronic production ability of cAMP, and thetherapeutic effect of cAMP is exhibited through CREB.

Experiment 7. In Vivo Test Using Convulsion as Index

A methylmalonic acid-administered mouse was used as a simplifiedneuropathy model for methylmalonic acidemia. In regard to this model,convulsion caused by administration of methylmalonic acid was used as anindex, and an evaluation of the candidate drug was carried out.

1. 1 mg/kg/dose 1 of a methylmalonic acid solution was subcutaneouslyadministered to four-week old C57BL/6J mice once a day, and evaluationwas performed in the sixth week of age.

2. Subcutaneous administration of db-cAMP (30 mg/kg/dose) and a PDE4inhibitor (1 mg/kg/day) as candidate drugs was performed, and thenumbers of times of convulsive seizure and the total durations beforeand after the administration were measured. Thus, an investigation ofcomparison was carried out. Meanwhile, before the administration,measurement as a control was carried out for 30 minutes, and after theadministration, measurement was carried out three times for 30 minuteseach time, from 15 minutes to 45 minutes, from 55 minutes to 85 minutes,and from 90 minutes to 120 minutes.

3. The results are presented in FIGS. 7 and 8.

(1) The number of times of seizure was decreased in all of themeasurement sections, through administration of the candidate drugs.

(2) Similarly, the total duration was shortened in all of themeasurement sections, through administration of the candidate drugs.

4. From these results, it was found that even in an in vivo condition,neuropathy in methylmalonic acidemia is suppressed by promoting anincrease in the cAMP concentration.

Experiment 8. In Vivo Test Using Survival Rate as Index

An evaluation of administration of candidate drugs was carried out usingthe survival rate as an index, using a lethal methylmalonic acid seizuremodel.

1. A 500 μM methylmalonic acid solution was intraperitoneallyadministered to eight-week old C57BL/6J mice at a dose of 20 ml/kg, andthus a lethal methylmalonic acid seizure model was produced.

2. After one day, after two days, and after three days from theadministration of methylmalonic acid, subcutaneous administration ofdb-cAMP (10 μg/kg/dose) as a candidate drug was performed, and acomparison of the survival rates in an administered group and anon-administered group was carried out.

3. The results are presented in FIG. 9.

(1) In the non-administered group, the survival rate was 35.3% one dayafter the administration of methylmalonic acid, and the survival ratewas 14.7% after two days.

(2) On the other hand, in the administered group, the survival rate was85.0% one day after the administration of methylmalonic acid, and thesurvival rate was 75.0% after two days. The administered group exhibiteda significantly high survival rate compared to the non-administeredgroup (p<0.001).

4. From these results, it was found that the vital prognosis based onseizure originating from methylmalonic acidemia is markedly amelioratedby promoting the increase in the cAMP concentration through theadministration of db-cAMP.

1. A therapeutic drug for neuropathy, treating or preventing neuropathyby increasing cAMP in organic acidemia.
 2. The therapeutic drug forneuropathy according to claim 1, further activating CREB.
 3. Thetherapeutic drug for neuropathy according to claim 1, wherein theorganic acidemia lowers the activity of Adenylate cyclase.
 4. Thetherapeutic drug for neuropathy according to claim 1, wherein theorganic acidemia is selected from any one or a plurality ofmethylmalonic acidemia and propionic acidemia.
 5. The therapeutic drugfor neuropathy according to claim 1, wherein the increase in cAMP isachieved by any one of Forskolin, GW9508, NECA, SKF77434, nicotinamide,dobutamine, and db-cAMP.
 6. The therapeutic drug for neuropathyaccording to claim 3, wherein the increase in cAMP is achieved bysuppressing or preventing a decrease in the activity of Adenylatecyclase.
 7. The therapeutic drug for neuropathy according to claim 1,wherein a life prolongation effect is increased by treating orpreventing neuropathy.
 8. A method for treating or preventing neuropathyin organic acidemia by using the therapeutic drug for neuropathyaccording to claim 1.